Hepatitis C virus (HCV) remodels hepatocyte endoplasmic reticulum (ER) membranes to establish protected sites of replication. These structures are thought to shield the viral RNAs from cytosolic cellular nucleases and innate immune sensors. The mechanism of cellular membrane reorganization is poorly understood. Although the viral NS4B protein has been historically linked to this function, more recent evidence points to a primary role for NS5A in membrane remodeling. We, and others, previously identified the cellular lipid kinase phosphatidylinositol-4 kinase-III? (PI4KA) as a requirement or HCV replication. We found that the viral NS5A protein binds PI4KA, recruits it to sites of viral replication, and stimulates its activity. In cells deficient for PI4KA expression, the HCV replicas collapses, resulting in aggregates of replicase that are non-functional. The enzymatic activity of PI4KA is required for HCV infection, suggesting a role for its product, PI(4)P. The exact function of PI4KA and PI(4)P in HCV replication is unclear but it has been suggested by play a role in recruiting proteins with PI(4)P binding domains, such as rab GTPases and lipid transfer proteins. These proteins may modify the lipid micro-environment of the ER to facilitate its remodeling. Additionally, the PI4KA-NS5A interaction is important for regulating the phosphorylation of NS5A, which is critical to NS5A function in HCV replication. While characterizing the role of the NS5A-PI4KA interaction in HCV replication, we hypothesized that an exciting class of HCV drugs currently entering phase III clinical trials called NS5A direct-acting antivirals (DAAs) might target this interaction. The mechanism of action for this drug class is unknown; however, it is thought to target the HCV NS5A protein since drug resistant mutations accumulate in the viral NS5A gene [1]. Indeed, we found that a prototype NS5A DAA, BMS-790052 (BMS), prevents the stimulation of PI4KA activity by HCV. Additionally, BMS produces a phenotype similar to silencing PI4KA in that the viral replicase collapses and aggregates. BMS does not inhibit PI4KA directly, nor does it inhibit the interaction of NS5A with PI4KA. It apparently prevents the activation of PI4KA by NS5A after initial binding. We propose to characterize the function of the NS5A-PI4KA interaction in establishing protected sites of replication while, in parallel, characterizing the mechanism of action of NS5A DAAs. This approach is logical given that these aims overlap biologically and that the NS5A DAAs will be an invaluable tool in studying NS5A- PI4KA function. The specific aims are: 1. Define the function of PI4KA in modifying the lipid microenvironment for HCV replication. 2. Define the role of PI4KA in regulating NS5A phosphorylation and function. 3. Define the mechanism of action of a prototype NS5A DAA in modulating the NS5A-PI4KA interaction.